Underivatized, aqueous soluble beta (1-3) glucan, composition and method of making same

ABSTRACT

The present invention relates to neutral, aqueous soluble β-glucans which exert potent and specific immunological effects without stimulating the production of certain cytokines, to preparations containing the novel β-glucans, and to a novel manufacturing process therefor. The neutral, aqueous soluble β-glucan preparation has a high affinity for the β-glucan receptor of human monocytes and retains two primary biological (or immunological) activities, (1) the enhancement of microbicidal activity of phagocytic cells, and (2) monocyte, neutrophil and platelet hemopoietic activity. Unlike soluble glucans described in the prior art, the neutral, aqueous soluble β-glucan of this invention neither induces nor primes IL-1β, and TNFα production in vitro and in vivo. Safe and efficacious preparations of neutral, aqueous soluble β-glucan of the present invention can be used in therapeutic and/or prophylactic treatment regimens of humans and animals to enhance their immune response, without stimulating the production of certain biochemical mediators (e.g., IL-1β, TNFα) that can cause detrimental side effects, such as fever and inflammation.

BACKGROUND OF THE INVENTION

[0001] In the early 1960's, zymosan, a crude insoluble yeast extractprepared by boiling yeast before and after trypsin treatment, was notedto produce marked hyperplasia and functional stimulation of theraticuloendothelial system in rodents. In animal studies; zymosanpreparations were shown to inactivate complement component C3, toenhance antibody formation, to promote survival following irradiation,to increase resistance to bacterial infections, to inhibit tumordevelopment, to promote graft rejection, and to inhibit dietary-inducedhypercholesterolemia and cholesterosis. Zymosan was shown to consist ofpolysacoharides, proteins, fats, and inorganic elements; however,subsequent studies identified the active components of the yeast cellwall as a pure polysaccharide, specifically β-glucan. In conventionalnomenclature, the polysaccharide β-glucan is known aspoly-(1-6)-β-O-glucopyranosyl-(1-3)-β-D-glucopyranose (PGG). Repetitionof biological assays with β-glucan indicated that most of the abovefunctional activities identified with zymosan were retained by thepurified β-glucan preparation.

[0002] The properties of β-glucan are quite similar to those ofendotoxin in increasing nonspecific immunity and resistance toinfection. The activities of β-glucan as an immune adjuvant andhemopoietic stimulator compare to those of more complex biologicalresponse modifiers (BRMs), such as bacillus Calmette-Guerin (BCG) andCorynebacterium parvum. The functional activities of yeast β-glucan arealso comparable to those structurally similar carbohydrate polymersisolated from fungi and plants. These higher molecular weight(1-3)-β-D-glucans such as schizophyllan, lentinan, krestin, grifolan,and pachyman exhibit similar immunomodulatory activities. A commonmechanism shared by all these β-glucan preparations is their stimulationof cytokines such as interleukin-1 (IL-1) and tumor necrosis factor(TNF). Lentinan has been extensively investigated for its antitumorproperties, both in animal models at 1 mg/kg for 10 days and in clinicaltrials since the late 1970's in Japan for advanced or recurrentmalignant lymphoma and colorectal, mammary, lung and gastric cancers. Incancer chemotherapy, lentinan has been administered at 0.5-5 mg/day,intramuscularly (I. M.) or intravenously (I. V.), two or three times perweek alone, or in combination with antineoplastic drugs. In addition tothe activities ascribed to yeast glucans, studies suggest lentinan actsas a T-cell immunopotentiator, inducing cytotoxic activities, includingproduction of interleukins 1 and 3 and colony-stimulating factors (CSF).(Chihara et al., 1989, Int. J. Immunotherapy, 4:145-154; Hamuro andChihara, In Lentinan, An Immunopotentiator)

[0003] Various preparations of both particulate and soluble β-glucanshave been tested in animal models to evaluate biological activities. Theuse of soluble and insoluble β-glucans alone or as vaccine adjuvants forviral and bacterial antigens has been shown in animal models to markedlyincrease resistance to a variety of bacterial, fungal, protozoan andviral infections. The hemopoietic effects of β-glucan have beencorrelated with increased peripheral blood leukocyte counts and bonemarrow and splenic cellularity, reflecting increased numbers ofgranulocyte-macrophage progenitor cells, splenic pluripotent stem cells,and erythroid progenitor cells, as well as, increased serum levels ofgranulocyte-monocyte colony-stimulating factor (GM-CSF). Furthermore,the hemopoietic and anti-infective effects of β-glucan were active incyclophosphamide-treated immunosuppressed animals. β-glucan was shown tobe beneficial in animal models of trauma, wound healing andtumorigenesis. However, various insoluble and soluble preparations ofβ-glucan differed significantly ink biological specificity and potency,with effective dosages varying from 25-to 500 mg/kg intravenously orintraperitoneally (I. P.) in models for protection against infection andfor hemopoiesis. Insoluble preparations demonstrated undesirabletoxicological properties manifested by hepatosplenomegaly and granulomaformation. Clinical interest was focused on a soluble glucan preparationwhich would retain biological activity yet yield negligible toxicitywhen administered systemically. Chronic systemic administration of asoluble phosphorylated glucan over a wide range of doses (40-1000 mg/kg)yielded negligible toxicity in animals (DiLuzio et al., 1979, lnt. J. ofCancer, 24:773-779; DiLuzio, U.S. Pat. No. 4,739,046).

[0004] The molecular mechanism of action of β-glucan has been elucidatedby the demonstration of specific β-glucan receptor binding sites on thecell membranes of human neutrophils and macrophages. Mannans, galactans,α(1-4)-linked glucose polymers and β(1-4)-linked glucose polymers haveno avidity for this receptor. These β-glucan binding sites areopsonin-independent phagocytic receptors for particulate activators ofthe alternate complement pathway, similar to Escherichia colilipopolysaccharide (LPS) and some animal red blood cells. Ligand bindingto the β-glucan receptor, in the absence of antibody, results incomplement activation, phagocytosis, lysosomal enzyme release, andprostaglandin, thromboxane and leukotriene generation; therebyincreasing nonspecific resistance to infection. However, solubleβ-glucan preparations described in the prior art demonstratedstimulation of cytokines. Increases in plasma and splenic levels ofinterleukins 1 and 2 (IL-1, IL-2) in addition to TNF were observed invivo and corresponded to induction of the synthesis of these cytokinesin vitro. (See Sherwood et al., 1987, Int. J. Immunopharmac., 9:261-267(enhancement of IL-1 and IL-2 levels in rats injected with solubleglucan); Williams et al., 1988, Int. J. Immunopharmac., 10:405-414(systemic administration of soluble glucan to AIDS patients increasedIL-1 and IL-2 levels which were accompanied by chills and fever);Browder et al., 1990, Ann. Sure, 211:605-613 (glucan administration totrauma patients increased serum IL-1 levels, but not TNF levels); Adachiet al., 1990, Chem. Pharm. Bull., 38:988-992 (chemically cross-linkedβ(1-3) glucans induced IL-1 production in mice).)

[0005] Interleukin-1 is a primary immunologic mediator involved incellular defense mechanisms. Numerous studies have been carried out onthe application of IL-1 to enhance non-specific resistance to infectionin a variety of clinical states. Pomposelli et al., J. Parent. Ent.Nutr. 12(2):212-218, (1988). The major problem associated with theexcessive stimulation or exogenous administration of IL-1 and othercellular mediators in humans is toxicity and side effects resulting fromthe disruption of the gentle balance of the immunoregulatory network.Fauci et al., Ann. Int. Med., 106:421-433 (1987). IL-1 is aninflammatory cytokine that has been shown to adversely affect a varietyof tissues and organs. For instance, recombinant IL-1 has been shown tocause death, hypotensive shock, leukopenia, thrombocytopenia, anemia andlactic acidosis. In addition, IL-1 induces sodium excretion, anorexia,slow wave sleep, bone resorption, decreased pain threshold andexpression of many inflammatory-associated cytokines. It is also toxicto the insulin secreting beta cells of the pancreas. Patients sufferingfrom a number of inflammatory diseases have elevated levels of IL-1 intheir systems. Administration of agents that enhance further IL-1production only exacerbate these inflammatory conditions.

[0006] Tumor necrosis factor is also involved in infection, inflammationand cancer. Small amounts of TNF release growth factors while in largeramounts, TNF can cause septic shock, aches, pains, fever, clotting ofblood, degradation of bone and stimulation of white blood cells andother immune defenses.

SUMMARY OF THE INVENTION

[0007] The present invention relates to neutral soluble β-glucans whichenhance a host's immune defense mechanisms to infection but do notinduce an inflammatory response, to preparations containing the neutralsoluble β-glucans, and to a novel manufacturing process therefor. In thepresent method, soluble glucan which induces cytokine production isprocessed through a unique series of acid, alkaline and neutraltreatment steps to yield a conformationally pure neutral soluble glucanpreparation with unique biological properties. The neutral solubleglucan preparation retains a specific subset of immunological propertiescommon to β-glucans but uniquely does not induce the production of IL-1and TNF in vitro or in vivo. Throughout this specification, unlessotherwise indicated, the expressions “neutral soluble glucan” and“neutral soluble β-glucan” refer to the composition prepared asdescribed in Example 1.

[0008] The neutral soluble glucan preparation is produced by treatinginsoluble glucan with acid to produce a water soluble glucan,dissociating the native conformations of the soluble glucan at alkalinepH, purifying the desired molecular weight fraction at alkaline pH,re-annealing the dissociated glucan fraction under controlled conditionsof time, temperature and pH to form a unique triple helicaalconformation, and further purifying under neutral pH to remove singlehelix and aggregated materials to yield a conformationally pure,neutral, water soluble, underivatized glucan which has a uniquebiological profile.

[0009] The neutral soluble glucan preparation has a high affinity forthe β-glucan receptor of human monocytes and retains two primarybiological activities, (1) the enhancement of microbicidal activity ofphagocytic cells, and (2) monocyte, neutrophil and platelet hemopoieticactivity. Unlike soluble glucans described in the prior art, the neutralsoluble glucan of this invention neither induces nor primes mononuclearcells to increase IL-1 and TNF production in vitro and in vivo.

[0010] The neutral soluble glucan preparation is appropriate forparenteral (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), topical, oral or intranasal administration to humans andanimals as an anti-infective to combat infection associated with burns,surgery, chemotherapy, bone marrow disorders and other conditions inwhich the immune system may be compromised. Neutral soluble glucanproduced by the present method can be maintained in a clear solution andequilibrated in a pharmaceutically acceptable carrier. Safe andefficacious preparations of the neutral soluble glucan of the presentinvention can be used in therapeutic and/or prophylactic treatmentregimens of humans and animals to enhance their immune response, withoutstimulating the production of certain biochemical mediators (e.g., IL-1and TNF) that can cause detrimental side effects, such as fever andinflammation.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 shows the general structure of neutral soluble glucan asbeing a linear β(1-3)-linked glucose polymer having periodic branchingvia a single β(1-6)-linked glucose moiety.

[0012]FIG. 2 shows a gel permeation chromatogram (pH 7) of solubleglucan which has not been purified by alkali dissociation andre-annealing. The chromatogram shows three species, referred to hereinas high molecular weight aggregate (Ag), Peak A and Peak B (single helixglucan).

[0013]FIG. 3 is a chromatogram obtained for the neutral soluble glucanby gel permeation chromatography. The solid line represents the neutralsoluble glucan at pH 7 and the broken line represents the neutralsoluble glucan at pH 13.

[0014]FIG. 4 is a chromatogram obtained for the single helix β-glucan(Peak B) by gel permeation chromatography. The solid line representsPeak B at pH 7 and the broken line represents Peak B at pH 13.

[0015]FIG. 5 shows the change in serum IL-1 levels, over time, takenfrom patients intravenously infused with placebo (broken line) orneutral soluble glucan (solid line).

[0016]FIG. 6 shows the change in serum TNF levels, over time, taken frompatients intravenously infused with lacebo (broken line) or neutralsoluble glucan (solid line).

[0017]FIG. 7 is a diagram representing peripheral blood counts fromirradiated mice following administration of neutral soluble glucan.

[0018]FIG. 8 is a diagram representing platelet cell counts fromcisplatin-treated mice following administration of neutral solubleglucan.

DETAILED DESCRIPTION OF INVENTION

[0019] The invention relates to a neutral soluble β-glucan polymer thatcan bind to the β-glucan receptor and activate only a desired subset ofimmune responses. The terms “neutral soluble β-glucan” and “neutralsoluble glucan”, unless otherwise specified, refer to the compositionprepared as described in Example 1.

[0020] This neutral soluble β-glucan has been shown to increase thenumber of neutrophils and monocytes as well as their direct infectionfighting activity (phagocytosis and microbial killing). However, theneutral soluble β-glucan does not stimulate the production ofbiochemical mediators, such as IL-1 and TNF, that can cause detrimentalside effects such as high fever, inflammation, wasting disease and organfailure. These advantageous properties make neutral soluble glucanpreparations of this invention useful in the prevention and treatment ofinfection because they selectively activate only those components of theimmune system responsible for the initial response to infection, withoutstimulating the release of certain biochemical mediators that can causeadverse side effects. The solution containing the neutral solubleβ-glucan also lacks the toxicity common to many immunomodulators.

[0021] The neutral soluble β-glucans of this invention are composed ofglucose monomers organized as a β(1-3) linked glucopyranose backbonewith periodic branching via β(1-6) glycosidic linkages. The neutralsoluble glucan preparations contain glucans, which have not beensubstantially modified by substitution with functional (e.g., charged)groups or other covalent attachments. The general structure of theneutral soluble glucan is shown in FIG. 1. The biologically activepreparation of this invention is a conformationally purified form ofβ-glucan produced by dissociating the native glucan conformations andre-annealing and purifying the resulting unique triple helicalconformation. The unique conformation of the neutral soluble glucancontributes to the glucan's ability to selectively activate the immunesystem without stimulating the production of detrimental biochemicalmediators.

[0022] The neutral soluble glucan preparations of this invention areprepared from insoluble glucan particles, preferably derived from yeastorganisms. See Manners et al., Biochem. J., 135:19-30, (1973) for ageneral procedure to make insoluble yeast glucans. Glucan particleswhich are particularly useful as starting materials in the presentinvention are whole glucan particles (WGP) described by Jamas et al., inU.S. Pat. Nos. 4,810,646, 4,992,540, 5,082,936 and 5,028,703, theteachings of all of which are hereby incorporated herein by reference.The source of the whole glucan particles can be the broad spectrum ofglucan-containing yeast organisms which contain β-glucans in their cellwalls. Whole glucan particles obtained from the strains Saccharomycescerevisiae R4 (NRRL Y-15903; deposit made in connection with U.S. Pat.No. 4,810,646) and R4 Ad (ATCC No. 74181) are particularly useful. Otherstrains of yeast that can be used include Saccharomyces delbrueckii,Saccharomyces rosei, Saccharomyces microellinsodes, Saccharomycescarlsbercensis, Schizosacharomyces pombe, Kluyveromycies lactis,Kluyveromyces fragilis, Kluyveromyces polysporus, Candida albicans,Candida cloacae, Candida troipicalis, Candida utilis, Hansenula wingeri,Hansenula arni, Hansenula henricii, Hansenula americana.

[0023] A procedure for extraction of whole glucan particles is describedby Jamas et al., in U.S. Pat. Nos. 4,810,646, 4,992,540, 5,082,936 and5,028,703. For the purpose of this present invention, it is notnecessary to conduct the final organic extraction and wash stepsdescribed by Jamas et al.

[0024] In the present process, whole glucan particles are suspended inan acid solution under conditions sufficient to dissolve theacid-soluble glucan portion. For most glucans, an acid solution having apH of from about 1 to about 5 and at a temperature of from about 20 toabout 100° C. is sufficient. Preferably, the acid used is an organicacid capable of dissolving the acid-soluble glucan portion. Acetic acid,at concentrations of from about 0.1 to about 5 M or formic acid atconcentrations of from about 50% to 98% (w/v) are useful for thispurpose. The treatment time may vary from about 10 minutes to about 20hours depending on the acid concentration, temperature and source ofwhole glucan particles. For example, modified glucans having more (1-6)branching than naturally-occurring, or wild-type glucans, require morestringent conditions, i.e., longer exposure times and highertemperatures. This acid-treatment step can be repeated under similar orvariable conditions. One preferred processing method is described in theexemplification using glucan derived from S. cerevisiae strain R4 Ad. Inanother embodiment of the present method, whole glucan particles fromthe strain, S. cerevisiae R4, which have a higher level of β(1-6)branching than naturally-occurring glucans, are used, and treatment iscarried out with 90% (w/v) formic acid at 20° C. for about 20 minutesand then at 85° C. for about 30 minutes.

[0025] The insoluble glucan particles are then separated from thesolution by an appropriate separation technique, for example, bycentrifugation or filtration. The pH of the resulting slurry is adjustedwith an alkaline compound such as sodium hydroxide, to a pH of about 7to about 14. The precipitate is collected by centrifugation and isboiled in purified water (e.g., USP) for three hours. The slurry is thenresuspended in hot alkali having a concentration sufficient tosolubilize the glucan polymers. Alkaline compounds which can be used inthis step include alkali-metal or alkali-earth metal hydroxides, such assodium hydroxide or potassium hydroxide, having a concentration of fromabout 0.01 to about 10 N. This step can be conducted at a temperature offrom about 40° C. to about 121° C., preferably from about 20° C. toabout 100° C. In one embodiment of the process, the conditions utilizedare a 1 M solution of sodium hydroxide at a temperature of about 80-100°C. and a contact time of approximately 1-2 hours. The resulting mixturecontains solubilized glucan molecules and particulate glucan residue andgenerally has a dark brown color due to oxidation of contaminatingproteins and sugars. The particulate residue is removed from the mixtureby an appropriate separation technique, e.g., centrifugation and/orfiltration. In another embodiment of the process the acid-solubleglucans are precipitated after the preceding acid hydrolysis reaction bythe addition of about 1.5 volumes of ethanol. The mixture is chilled toabout 4° C. for two (2) hours and the resulting precipitate is collectedby centrifugation or filtration and washed with water. The pellet isthen resuspended in water, and stirred for three (3) to twelve (12)hours at a temperature between about 20° C. and 100° C. At this pointthe pH is adjusted to approximately 10 to 13 with a base such as sodiumhydroxide.

[0026] The resulting solution contains dissociated soluble glucanmolecules. This solution is now purified to remove traces of insolubleglucan and high molecular weight soluble glucans which can causeaggregation. This step can be carried out by an appropriate purificationtechnique, for example, by ultrafiltration, utilizing membranes withnominal molecular weight (NMW) levels or cut-offs in the range of about1,000 to 100,000 daltons. It was discovered that in order to preventgradual aggregation or precipitation of the glucan polymers thepreferred membrane for this step has a nominal molecular weight cut-offof about 100,000 daltons. The soluble glucan is then further purified atalkaline pH to remove low molecular weight materials. This step can becarried out by an appropriate purification technique, for example, byultrafiltration, utilizing membranes with nominal molecular weightlevels or cut-offs in the range of 1,000 to 30,000 daltons.

[0027] The resulting dissociated soluble glucan is re-annealed undercontrolled conditions of time (e.g., from about 10 to about 120minutes), temperature (e.g., from about 50 to about 70° C.) and pH. ThepH of the solution is adjusted to the range of about 3.5-11 (preferably6-8) with an acid, such as hydrochloric acid. The purpose of thisre-annealing step is to cause the soluble glucan to rearrange from asingle helix conformation to a new ordered triple helical conformation.The re-annealed glucan solution is then size fractionated, for exampleby using 30,000-70,000 NMW and 100,000-500,000 NMW cut-off membraneultrafilters to selectively remove high and low molecular weight solubleglucans. Prior to sizing, the soluble glucans exist as a mixture ofconformations including random coils, gel matrices or aggregates, triplehelices and single helices. The objective of the sizing step is toobtain an enriched fraction for the re-annealed conformation of specificmolecular weight. The order in which the ultrafilters are used is amatter of preference.

[0028] The concentrated fraction obtained is enriched in the soluble,biologically active neutral soluble glucan. The glucan concentrate isfurther purified, for example, by diafiltration using a 10,000 daltonmembrane. The preferred concentration of the soluble glucan after thisstep is from about 2 to about 10 mg/ml.

[0029] The neutralized solution can then be further purified, forexample, by diafiltration, using a pharmaceutically acceptable medium(e.g., sterile water for injection, phosphate-buffered saline (PBS) ,isotonic saline, dextrose) suitable for parenteral administration. Thepreferred membrane for this diafiltration step has a nominal molecularweight cut-off of about 10,000 daltons. The final concentration of theglucan solution is adjusted in the range of about 0. 5 to 10 mg/ml. Inaccordance with pharmaceutical manufacturing standards for parenteralproducts, the solution can be terminally sterilized by filtrationthrough a 0.22 μm filter. The neutral soluble glucan preparationobtained by this process is sterile, non-antigenic, essentiallypyrogen-free, and can be stored at room temperature (e.g., 15-30° C.)for extended periods of time without degradation. This process is uniquein that it results in a neutral aqueous solution of (pH 4.5 to 7.0)immunologically active glucans which is suitable for parenteraladministration.

[0030] For purposes of the present invention, the term “soluble” as usedherein to describe glucans obtained by the present process, means avisually clear solution can be formed in an aqueous medium such aswater, PPBS, isotonic saline, or a dextrose solution having a neutral pH(e.g., from about pH 5 to about 7.5), at room temperature (about 20-25°C.) and at a concentration of up to about 10 mg/ml. The term “aqueousmedium” refers to water and water-rich phases, particularly topharmaceutically acceptable aqueous liquids, including PBS, saline anddextrose solutions. The expression “visually clear” means that at aconcentration of a mg/ml, the absorption of the solution at 530 nm isless than OD 0.01 greater than the OD of an otherwise identical solutionlacking the B-glucan component.

[0031] The resulting solution is substantially free of proteincontamination, is non-antigenic, non-pyrogenic and is pharmaceuticallyacceptable for parenteral administration to animals and humans. However,if desired, the soluble glucan can be dried by an appropriate dryingmethod, such as lyophilization, and stored in dry form.

[0032] The neutral soluble glucans of this invention can be used assafe, effective, therapeutic and/or prophylactic agents, either alone oras adjuvants, to enhance the immune response in humans and animals.Soluble glucans produced by the present method selectively activate onlythose components that are responsible for the initial response toinfection, without stimulating or priming the immune system to releasecertain biochemical mediators (e.g., IL-1, TNF, IL-6, IL-8 and GM-CSF)that can cause adverse side effects. As such, the present soluble glucancomposition can be used to prevent or treat infectious diseases, inmalnourished patients, patients undergoing surgery and bone marrowtransplants, patients undergoing chemotherapy or radiotherapy,neutropenic patients, HIV-infected patients, trauma patients, burnpatients, patients with chronic or resistant infections such as thoseresulting from myeiodysplastic syndrome, and the elderly, all of who mayhave weakened immune systems. An immunocompromised individual isgenerally defined as a person who exhibits an attenuated or reducedability to mount, a normal cellular and/or humoral defense to challengeby infectious agents, e.g., viruses, bacteria, fungi and protozoa. Aprotein malnourished individual is generally defined as a person who hasa serum albumin level of less than about 3.2 grams per deciliter (g/dl)and/or unintentional weight loss of greater than 10% of usual bodyweight.

[0033] More particularly, the method of the invention can be used totherapeutically or prophylactically treat animals or humans who are at aheightened risk of infection due to imminent surgery, injury, illness,radiation or chemotherapy, or other condition which deleteriouslyaffects the immune system. The method is useful to treat patients whohave a disease or disorder which causes the normal metabolic immuneresponse to be reduced or depressed, such as HIV infection (AIDS). Forexample, the method can be used to pre-initiate the metabolic immuneresponse in patients who are undergoing chemotherapy or radiationtherapy, or who are at a heightened risk for developing secondaryinfections or post-operative complications because of a disease,disorder or treatment resulting in a reduced ability to mobilize thebody's normal metabolic responses to infection. Treatment with theneutral soluble glucans has been shown to be particularly effective inmobilizing the host's normal immune defenses, thereby engendering ameasure of protection from infection in the treated host.

[0034] The present-composition is generally administered to an animal ora human in an amount sufficient to produce immune system enhancement.The mode of administration of the neutral soluble glucan can be oral,enteral, parenteral, intravenous, subcutaneous, intraperitoneal,intramuscular, topical or intranasal. The form in which the compositionwill be administered (e.g., powder, tablet, capsule, solution, emulsion)will depend upon the route by which it is administered. The quantity ofthe composition to be administered will be determined on an individualbasis, and will be based at least in part on consideration of theseverity of infection or injury in the patient, the patient's conditionor overall health, the patient's weight and the time available beforesurgery, chemotherapy or other high-risk treatment. In general, a singledose will preferably contain approximately 0.01 to approximately 10 mgof modified glucan per kilogram of body weight, and preferably fromabout 0.1 to 2.5 mg/kg. The dosage for topical application will dependupon the particular wound to be treated, the degree of infection andseverity of the wound. A typical dosage for wounds will be from about0.001 mg/ml to about 2 mg/ml, and preferably from about 0.01 to about0.5 mg/ml.

[0035] In general, the compositions of the present invention can beadministered to an individual periodically as necessary to stimulate theindividual's immune response. An individual skilled in the medical artswill be able to determine the length of time during which thecomposition is administered and the dosage, depending upon the physicalcondition of the patient and the disease or disorder being treated. Asstated above, the composition may also be used as a preventativetreatment to preinitiate the normal metabolic defenses which the bodymobilizes against infections.

[0036] Neutral soluble β-glucan can be used for the prevention andtreatment of infections caused by a broad spectrum of bacterial, fungal,viral and protozoan pathogens. The prophylactic administration ofneutral soluble β-glucan to a person undergoing surgery, eitherpreoperatively, intraoperatively and/or post-operatively, will reducethe incidence and severity of post-operative infections in both normaland high-risk patients. For example, in patients undergoing surgicalprocedures that are classified as contaminated or potentiallycontaminated (e.g., gastrointestinal surgery, hysterectomy, cesareansection, transurethral prostatectomy) and in patients in whom infectionat the operative site would present a serious risk (e.g., prostheticarthroplasty, cardiovascular surgery), concurrent initial therapy withan appropriate antibacterial agent and the present neutral solubleglucan preparation will reduce the incidence and severity of infectiouscomplications.

[0037] In patients who are immunosappressed, not only by disease (e.g.,cancer, AIDS) but by courses of chemotherapy and/or radiotherapy, theprophylactic administration of the soluble glucan will reduce theincidence of infections caused by a broad spectrum of opportunisticpathogens including many unusual bacteria, fungi and viruses. Therapyusing neutral soluble β-glucan has demonstrated a significantradio-protective effect with its ability to enhance and prolongmacrophage function and regeneration and, as a result enhance resistanceto microbial invasion and infection.

[0038] In high risk patients (e.g., over age 65, diabetics, patientshaving cancer, malnutrition, renal disease, emphysema, dehydration,restricted mobility, etc.) hospitalization frequently is associated witha high incidence of serious nosocomial infection. Treatment with neutralsoluble β-glucan may be started empirically before catheterization, useof respirators, drainage tubes, intensive care units, prolongedhospitalizations, etc. to help prevent the infections that are commonlyassociated with these procedures. Concurrent therapy with antimicrobialagents and the neutral soluble β-glucan is indicated for the treatmentof chronic, severe, refractory, complex and difficult to treatinfections.

[0039] The compositions administered in the method of the presentinvention can optionally include other components, in addition to theneutral soluble β-glucan. The other components that can be included in aparticular composition are determined primarily by the manner in whichthe composition is to be administered. For example, a composition to beadministered orally in tablet form can include, in addition to neutralsoluble β-glucan, a filler (e.g., lactose), a binder (e.g.,carboxymethyl cellulose, gum arabic, gelatin), an adjuvant, a flavoringagent, a coloring agent and a coating material (e.g., wax orplasticizer). A composition to be administered in liquid form caninclude neutral soluble β-glucan and, optionally, an emulsifying agent,a flavoring agent and/or a coloring agent. A composition for parenteraladministration can be mixed, dissolved or emulsified in water, sterilesaline, PBS, dextrose or other biologically acceptable carrier. Acomposition for topical admihistration can be formulated into a gel,ointment, lotion, cream or other form in which the composition iscapable of coating the site to be treated, e.g., wound site.

[0040] Compositions comprising neutral soluble glucan can also beadministered topically to a wound site to stimulate and enhance woundhealing and repair. Wounds due to ulcers, acne, viral infections, fungalinfections or periodontal disease, among others, can be treatedaccording to the methods of this invention to accelerate the healingprocess. Alternatively, the neutral soluble β-glucan can be infectedinto the wound or afflicted area. In addition to wound repair, thecomposition can be used to treat infection associated therewith or thecausative agents that result in the wound. A composition for topicaladministration can be formulated into a gel, ointment, lotion, cream orother form in which the composition is capable of coating the site to betreated, e.g., wound site. The dosage for topical application willdepend upon the particular wound to be treated, the degree of infectionand severity of the wound. A typical dosage for wounds will be fromabout 0.01 mg/ml to about 2 mg/ml, and preferably from about 0.01 toabout 0.5 mg/ml.

[0041] Another particular use of the compositions of this invention isfor the treatment of myeiodysplastic syndrome (IDS). MDS, frequentlyreferred to as preleukemia syndrome, is a group of clonal hematopoieticstem cell disorders characterized by abnormal bone marrowdifferentiation and maturation leading to peripheral cytopenia with highprobability of eventual leukemic conversion. Recurrent infection,hemorrhaging and terminal infection resulting in death typicallyaccompany MDS. Thus, in order to reduce the severity of the disease andthe frequency of infection, compositions comprising modified glucan canbe chronically administered to a patient diagnosed as having MDSaccording to the methods of this invention, in order to specificallyincrease the infection fighting activity of the patient's white bloodcalls. Other bone marrow disorders, such as a plastic anemia (acondition of quantitatively reduced and defective hematopciesis) can betreated to reduce infection and hemorrhage that are associated with thisdisease state.

[0042] Neutral soluble glucan produced by the present method enhancesthe non-specific defenses of mammalian mononuclear cells andsignificantly increases their ability to respond to an infectiouschallenge. The unique property of neutral soluble glucan macrophageactivation is that it does not result in increased body temperatures(i.e. fever) as has been reported with many non-specific stimulants ofthose defenses. This critical advantage of neutral soluble glucan maylie in the natural profile of responses it mediates in white bloodcells. It has been shown that the neutral soluble β-glucan of thepresent invention selectively activates immune responses but does notdirectly stimulate or prime cytokine (e.g., IL-1 and TNF) release frommononuclear cells, thus distinguishing the present neutral solubleglucan from other glucan preparations (e.g., lentinan, kresein) andimmunostimulants.

[0043] In addition, it has been-demonstrated herein that the neutralsoluble glucan preparation of the present invention possesses anunexpected platelet stimulating property. Although it was known thatglucans have the ability to stimulate white blood cell hematopoiesis,the disclosed platelet stimulating property had not been reported oranticipated. This property can be exploited in a therapeutic regimen foruse as an adjuvant in parallel with radiation or chemotherapy treatment.Radiation and chemotherapy are known to result in neutropenia (reducedpolymorphonuclear (PMN) leukocyte cell count) and thrombocytopenia(reduced platelet count). At present, these conditions are treated bythe administration of colony-stimulating factors such as GM-CSF andgranulocyte colony-stimulating factor (G-CSF). Such factors areeffective in overcoming neutropenia, but fail to impact uponthrombocytopenia. Thus, the platelet stimulating property of the neutralsoluble glucan preparation of this invention can be used, for example,as a therapeutic agent to prevent or minimize the development ofthrombocytopenia which limits the dose of the radiation orchemotherapeutic agent which is used to treat cancer.

[0044] The invention is further illustrated by the following Examples.

EXAMPLES Example 1: Preparation Of Neutral Soluble Glucan From S.Cerevisiae

[0045]Saccharomyces cerevisiae strain R4 Ad (a non-recombinantderivative of wild-type strain A364A), was grown in a large-scalefermentation culture using a defined glucose, ammonium sulfate minimalmedium. The production culture was maintained under glucose limitationin a feed-batch-mode (New Brunswick MPP80). When the growing culturereached late logarithmic phase, the fermentation was ended and theβ-glucan was stabilized by adjusting the culture to pH 12±0.5 using 10 MNaOH. The yeast cells containing β-glucan were harvested bycontinuous-flow centrifugation (Westfalia SA-1). After centrifugation,the cells were collected into a stainless steel extraction vessel.

[0046] The first step in the extraction process was an alkalineextraction accomplished by mixing the cells with 1 M sodium hydroxide(NaOH) at 90±5° C. for 1 hour. Upon completion of this alkalineextraction, the β-glucan remained in the solid phase, which wascollected by continuous centrifugation (Westfalia SA-1). The collectedcell wall fraction was extracted a second time using the same procedureand under the same conditions. Treatment with alkali hydrolyzed andsolubilized the cellular proteins, nucleic acids, mannans, solubleglucans and polar lipids into the supernatant fraction, anddeacety-lated chitin to chitosan in the cell wall.

[0047] The second step in the extraction process was a pH 4.5±10.05(adjusted with concentrated HCl) extraction at 75±0.5° C. for 1 hour.This was followed by a 0.1 M acetic acid extraction to complete theremoval of glycogen, chitin, chitosan and remaining proteins. The solidswere collected and rinsed twice with Purified Water USP to remove anyresidual acid as well as any yeast degradation products.

[0048] The third step in the extraction process was a set of six organicextractions. The first four extractions were carried out in isopropanol.The solids were collected by centrifugation and then subjected to twoacetone extractions. The two-stage organic extractions eliminatednonpolar lipids and hydrophobic proteins which may have co-purified withthe drug substance. The resulting wet solids were dried in a vacuum ovenat 65±5° C. for 48-96 hours to yield a free-flowing powder.

[0049] At this stage the extraction process yielded a stable, insolubleintermediate consisting of approximately 90% β-glucan, called wholeglucan particles (WGPs). The dry WGP intermediate was stored at 15-30°C. until further use.

[0050] The WGP powder was resuspended in 98% (w/v) formic acid, in aglass reaction vessel at room temperature. The resulting mixture washeated to 85±5° C. for 20 minutes. Under these conditions, the WGPs werepartially hydrolyzed and solubilized to provide the desired molecularweight distribution of soluble β-glucan which was then precipitated byadding 1.5 volumes of ethanol. After complete mixing, the preparationwas centrifuged to collect the β-glucan precipitate. Any residual formicacid was removed by boiling the β-glucan preparation in Purified WaterUSP for three hours.

[0051] Any unhydrolyzed WGPs were then removed from the β-glucansolution by centrifugation. The β-glucan solution was raised to pH12.5±0.5 by the addition of the concentrated sodium hydroxide. Theremaining purification steps were carried out by ultrafiltration.

[0052] The soluble alkaline β-glucan preparation was passed through a100,000 nominal molecular weight (NMW) cut-off membrane ultrafilter(Amicon DC10). Under alkaline conditions this membrane ultrafilterremoved insoluble and high. molecular weight soluble β-glucan. Trace lowmolecular weight degradation products were then removed by recirculationthrough a 10,000 NMW cut-off membrane ultrafilter. The ultrafiltrationwas conducted as a constant volume wash with 0.1 M NaOH.

[0053] The β-glucan solution was re-annealed under controlled conditionsby adjusting the pH to 7.0±0.5 with concentrated hydrochloric acid,heating to 60±10° C., which was maintained for 20 minutes and thencooled. The neutral re-annealed solution was then concentrated andwashed with Sodium Chloride Injection USP in a 70,000 NMW cut-offmembrane ultrafilter (Filtron Minisep) to enrich for the re-annealedneutral soluble glucan. Next the material was filtered through a 300,000NMW cut-off membrane ultrafilter (Filtron Minisep) to remove highmolecular weight and aggregated glucan molecules. In the sameultrrafilter, the neutral soluble glucan material was washed with SodiumChloride Injection USP in a constant volume wash mode.

[0054] The neutral soluble glucan was then concentrated in a 10,000 NMWcut-off membrane ultrafilter. The concentration process continued untila concentration of at least 1.0 mg/ml hexose equivalent was achieved.

[0055] The resulting neutral soluble glucan was then subjected tofiltration through a depyrogenating filter (0.1 micron Posidyne) and asterile 0.2 micron filter (Millipak) to yield sterile, pyrogen-freeneutral soluble glucan. The neutral soluble glucan solution was storedat controlled room temperature (15-30° C.) until further use. Theaqueous solubility of neutral soluble glucan in the pH range of 4 to 8is approximately 100 mg/ml. The solubility increased with increasing pHand reached approx. 150 mg/ml at pH 13.

Example 2: Analysis Of Neutral Soluble Glucan

[0056] A. Glucose, Mannose and Glucosanine

[0057] Monosaccharide analysis was performed to quantitate the relativeamounts of β-glucan (as glucose), mannan or phosphomannan (as mannose),and chitin (as N-acetyl glucosamine) in the neutral soluble glucan. Thesample was hydrolyzed to monosaccharides in 2 M trifluoroacetic acid for4 hours at 110° C., evaporated to dryness, and redissolved in water.Monosaccharides were separated on a Dionex HPLC system using a CarboPacPA100 column (4×250 mm) using 5 M NaOH at 1 ml/min and quantitated usinga pulsed electrochemical detector (Dionex Model PED-1). The sensitivityof this assay for monosaccharides is 0.1% (w/w).

[0058] Glucose (retention time of 16.6 mm) was identified as the onlymonosaccharide component of neutral soluble glucan along with traces ofglucose degradation products (from hydrolysis) anhydroglucose at 2.5 minand 5-hydroxymethylfurfural at 4.3 min. The results confirm that neutralsoluble glucan consisted of ≧98% glucose.

[0059] B. FTIR

[0060] Fourier transform infrared spectroscopy by diffuse reflectance(FTIR, Matson Instruments, Polaris) of lyophilized neutral solubleglucan samples was used to determine the anomeric structure (α vs. β),and linkage type (β(1-3), β(1-6), β(1-4)) present in neutral solubleglucan. Absorption maxima of 890 cm⁻¹ identified β(1-3) linkages; 920cm⁻¹ identified β(1-6) linkages. No presence of α-linked anomers (e.g.,glycogen, 850 cm⁻¹) or β(1-4)-linked polysaccharides (e.g., chitin, 930cm⁻¹) were detected.

Example 3: Conformational Analysis

[0061] A solution of β-glucan which was not processed by alkalidissociation and re-annealing was analyzed for its compositionalidentity by gel permeation chromatography (pH 7) and found to containmultiple species, referred to herein as high molecular weight aggregate(Ag), Peak A and Peak B (See FIG. 2). Neutral soluble glucan which wasprepared by alkali dissociation and re-annealing as described in Example1, is present as a single peak (see FIG. 3) with an average molecularweight of 92,660 daltons at pH 7. The distinct conformations of neutralsoluble glucan and Peak B were demonstrated by gel permeationchromatography at pH 7 and pH 13 using a refractive index detector.Neutral soluble glucan underwent a significant conformational transitionfrom pH 7 to pH 13 which illustrates complete dissociation of themultiple helix at pH 7 to a single helical form at pH 13 (see FIG. 3).In contrast, Peak B only underwent a slight shift in molecular weightfrom pH 7 to pH 13 (see FIG. 4). The molecular weight of neutral solubleglucan and Peak B glucans as a function of pH is shown below in Table 1.TABLE 1 MW MW Ratio Sample pH 7 pH 13 (pH 7/pH 13) Neutral soluble92,666 18,693 4.96 glucan Peak B 8,317 7,168 1.16

[0062] The conformation of neutral soluble glucan and Peak B glucan wasalso determined by aniline blue complexing (Evans et al., 1984, Carb.Pol., 4:215-230; Adachi et al., 1988, Carb. Res., 177: 91-100), usingcurdlan, a linear β(1-3) glucan, as the triple helix control andpustulan, a β(1-6) glucan, as a non-ordered conformational control. Theresults are discussed below and shown in Table 2.

[0063] The curdlan triple helix control complexed with aniline blueresulting in high fluorescence. Increasing the NaOH concentration beganto dissociate the curdlan triple helix slightly, but NaOHconcentrations >0.25 M are required for complete dissociation ofcurdlan. The pustulan non-ordered control only formed a weak complexwith aniline blue resulting in low fluorescence measurements which werenot affected by NaOH concentration.

[0064] The neutral soluble glucan complexed effectively with anilineblue at low NaOH concentration (25 mM NaOH) resulting in highfluorescence. However, the neutral soluble glucan conformationdissociated significantly (50%) at NaOH concentrations as low as 150 mMNaOH indicating that it exists as a unique conformation compared tonaturally occurring β-glucans, such as laminarin and curdlan, whichrequire significantly higher NaOH concentrations for dissociation tooccur. Peak B formed a weak complex with aniline blue due to its singlehelical conformation. TABLE 2 Conformational Analysis of Glucans byAniline Blue Complexing Fluorescence 25 mM 100 mM 150 mM Test MaterialNaOH NaOH NaOH Blank 0 2 0 Curdlan 53.5 41.6 36 β(1-3) glucan Pustulan9.8 8.3 8.0 β(1-6) glucan Neutral soluble glucan 40 25.6 20.2 Peak B12.4 6.2 4.1

Example 4: Effects Of Neutral Soluble Glucan On Human MonocyteProduction Of TNFα

[0065] Human peripheral blood mononuclear cells were isolated (Janusz etal., (1987), J. Immunol., 138: 3897-3901) from normal citrated anddextran-treated blood, washed in Hank's balanced salt solution (HBSS),lacking calcium, magnesium, and phenol red, and purified by gradientcentrifugation on cushions of Ficoll-Paque (Pharmacia Fine Chemicals,Piscataway, N.J.). The mononuclear cells were collected into HBSS,washed twice, resuspended in RPMI 1640 Medium (Gibco, Grand Island,N.Y.) containing 1% heat-inactivated autologous serum (56° C. for 30min.), and counted on the Coulter counter.

[0066] For the preparation of monocyte monolayers, 1 ml of 2.2×10⁶mononuclear cells/ml was plated into wells of 24-well tissue cultureplates (Costar, Cambridge, Mass.), incubated for 1 hour at 37° C. in ahumidified atmosphere of 5% CO₂, and washed three times with RPMI toremove nonadherent cells. A second 1 ml aliquot of 2.2×10⁶ mononuclearcells/ml was layered into each well and incubated for 2 hours describedabove before removal of the nonadherent cells. By visual enumeration at40X with an inverted phase microscope and a calibrated reticle, thenumber of adherent calls for 30 different donors was 0.77±0.20×10⁶ perwell (mean±SD). By morphology and nonspecific esterase staining, >95% ofthe adherent cells were monocytes.

[0067] Monocyte monolayers were incubated at 37° C. in the CO₂ chamberfor 0 to 8 hours with 0.5 ml of RPMI, 1% heat-inactivated autologousserum, 10 mM EPES, and 5 mM MgCl₂ in the absence and presence of variousglucan preparations. The culture supernatant was removed, clarified bycentrifugation at 14,000 g for 5 min at 4° C., and stored at −70° C.before assay of TNFα.

[0068] The concentration of TNFα in the monocyte supernatants wasmeasured by an enzyme-linked immunoadsorbent assay (ELISA) with theBIORINE TNF Test kit (T Cell Sciences, Cambridge, Mass.), which had alower limit of detectability of 40 pg/ml. The data are expressed as pgper 10⁶ monocytes, which was calculated by dividing the quantity ofcytokine in 0.5 ml of supernatant by the number of monocytes per well.

[0069] For the determination of cell-associated levels of TNFα, theadherent monocytes were lysed in 0.25 ml PBS by three rounds of freezingand thawing, the lysates were cleared of debris by centrifugation at14,000 g for 5 min at 4° C., and the resulting supernatants were storedat −70° C. Newly prepared monocyte monolayers contained no detectablelevels of intracellular TNFα.

[0070] The results are shown in Tables 3 and 4 below. TABLE 3 TNFαSynthesis by Human Monocytes Stimulated with Various Glucan PreparationsTNFα (pg/10⁶ monocytes) Glucan Conc. 1 2 3 Mean ± SD Buffer Control 3639 2 26 ± 21 Neutral soluble 1 mg/ml 44 51 33 43 ± 9 glucan Laminarin 1mg/ml 372 324 227 308 ± 74 Whole 4 × 10⁷/ml 2129 1478 1683 1763 ± 333Glucan particles

[0071] TABLE 4 TNFα Stimulation by Different Conformational Structuresof Soluble β-Glucan TNFα Glucan Conc. (pg/10⁶ monocytes) Buffer Control1 mg/ml 40 Laminarin 1 mg/ml 1312 Neutral soluble glucan 1 mg/ml 16 PeakB 1 mg/ml 1341 Glucan Particles 4 ± 10⁷/ml 2065

[0072] Table 3 shows that TNFα was stimulated by insoluble glucanparticles and by laminarin, a soluble β(1-6) and β(1-3) linked glucan.There was no stimulation of TNFα by neutral soluble glucan. Table 4shows similar results, but further confirms that TNFα stimulation isdependent upon conformational structure. The neutral soluble glucan didnot stimulate TNFα while Peak B (single helical conformation) didstimulate TNFα.

Example 5: Avidity Of Neutral Soluble Glucan For The Glucan Receptor

[0073] Monolayers of human monocytes, prepared on siliconized glasscoverslips (Czop et al., 1978, J. Immunol., 120:1132), were incubatedfor 18 minutes at 37° C. in a humidified 5% CO₂ incubator with either0.25 ml of buffer (RPMI-Mg-HEPES) or a range of concentrations (0.1-50μg/ml) of neutral soluble glucan. The monocyte monolayers were thenwashed twice with 50 ml of RPMI 1640 medium and were layered with 0.25ml of 4.8×10⁶/ml zymosan particles (Czop and Austen, 1985, J. Immunol.,134:2588-2593). After a 30 minute incubation at 37° C., the monolayerswere washed three times with 50 ml of Hank's balanced salt solution toremove noningested zymosan particles. The monolayers were then fixed andstained with Giemsa. The ingestion of zymosan particles by at least 300monocytes per monolayer was determined by visual observation under a1000X light microscope.

[0074] Monocyte monolayers protreated with buffer, 50 or 500 μg/ml ofneutral soluble glucan as described above were subsequently tested fortheir capacity to ingest IgG coated sheep erythrocytes (E′IgG). After an18 minute preincubation with the neutral soluble glucan, the monolayerswere incubated with 0.25 ml of 1×10⁷/ml E′IgG for 30 minutes at 37° C.,washed three times with 50 ml of Hank's balanced salt solution, treatedfor 4 minutes with 0.84% NH₄Cl to lyse noningested E′IgG, and fixed andstained as described above. The percentages of monocytes ingesting ≧1and ≧3 E′IgG were determined by counting at least 300 monocytes permonolayer.

[0075] The percent inhibition of monocyte ingestion was determined bysubtracting the percentage of monocytes ingesting targets afterpretreatment with the neutral soluble glucan from the percentageingesting targets after pretreatment with buffer, dividing this numberby the percentage ingesting targets after pretreatment with buffer andmultiplying by 100. The data are expressed as the mean of twoexperiments and are reported in Table 5. TABLE 5 Glucan-receptor BindingCapacity of Distinct Conformations of Soluble β-glucans Test MaterialConc. % Inhibition Buffer —  0% Neutral soluble glucan  50 μg/ml 74% 500μg/ml 86% Peak B  50 μg/ml 50% 500 μg/ml 56%

[0076] Both β-glucan preparations tested above inhibited monocyteingestion of zymosan particles demonstrating their capacity tocompetitively bind to the β-glucan receptor, on human monocytes. Neutralsoluble glucan demonstrated a higher receptor binding capacity than PeakB as indicated by the greater level of inhibition achieved at both 50μg/ml and 500 μg/ml. This biological assay demonstrates that the neutralsoluble glucan is a superior ligand for the β-glucan receptor.

Example 6: Lack Of In Vitro Stimulation Of IL-1βAnd TNFα From HumanMononuclear Cells

[0077] Venous blood was obtained from healthy male volunteers andmononuclear cells were fractionated by Ficoll-Hypacue centrifugation.The mononuclear cells were washed, resuspended in endotoxin-freeRPMI-1640 culture medium—ultrafiltered to remove endotoxins as describedelsewhere (Dinarello et al., 1987, J. Clin. Microbiol. 25:1233-8)—at aconcentration of 5×10⁶ cells/ml and were aliquoted into 96-wellmicrotiter plates (Endres et al., 1989, N. E. J. Med. 320:265-271). Thecells were then incubated with either 1 ng/ml endotoxin(lipopolysaccharide, E. coli 055:B5, Sigma, St. Louis), or 10 to 1000ng/ml βglucan, at 37° C. for 24 hours in 5% CO₂ and then lysed by threefreeze-thaw cycles (Endres et al., 1989, N. E. J. Med. 320:265-271).Synthesis of IL-1β, and TNFα was determined by specificradioimmunoassays as described elsewhere (List et al., 1987, Lymph Res.6:229-244; Lonnemann et al., 1988, Lymph. Res. 7:75-84; Van der Meer etal., 1988, J. Leukocyte Biol. 43:216-223.

[0078] To determine if neutral soluble glucan could act endotoxin, aknown cytokine stimulant, mononuclear cells were pre-incubated- with 1,10, and 1000 ng/ml of the neutral soluble glucan for 3 hours at 37° C.in 5% CO₂. The cells were washed to remove neutral soluble glucan andwere then incubated with 1 ng/ml endotoxin as described above. IL-1β andTNFα were determined as described above.

[0079] The results are summarized in Table 6. Neutral soluble glucanused as a stimulant at doses of 10-1000 ng/ml alone did not induceincreased levels of IL-1β or TNFα synthesis over the control buffertreated cells. Endotoxin LPS, a known stimulant, resulted insignificantly increased levels of both cytokines. In a second phase ofthis experiment neutral soluble glucan was tested for its ability to actas a priming agent for mononuclear cell cytokine synthesis. The cellsfrom the same donors were pre-incubated with three doses of neutralsoluble glucan (10-1000 ng/ml) and were then exposed to endotoxin as aco-stimulant. Neutral soluble glucan did not result in any amplificationof the IL-1β and TNFα levels compared to endotoxin alone. TABLE 6 InVitro IL-1β and TNFα Synthesis by Human Peripheral Blood MononuclearCells IL-1β TNFα Stimulant (ng/ml)¹ (ng/ml)¹ Cells only <0.10 0.14Neutral  10 ng/ml 0.13 0.16 soluble  100 ng/ml 0.12 0.16 glucan 1000ng/ml <0.10 0.14 LPS   1 ng/ml 2.62 2.22 LPS (1 ng/ml) +  10 ng/ml 2.622.25 Neutral  100 ng/ml 2.57 2.07 soluble 1000 ng/ml 2.85 2.27 glucan

Example 7: In Vivo Protection Against Infection In Mice

[0080] A sepsis model was developed in rats to characterize the efficacyof β-glucan in protecting an immunologically intact host against seriousinfections, such as those which commonly occur following abdominal..surgery. The rat model for intra-abdominal sepsis has been welldescribed in the scientific literature (Onderdonk et al., 1974, Infect.Imun., 10:1256-1259).

[0081] Groups of rats received neutral soluble glucan (100 μg/0.2 ml) orsaline control (0.2 ml) intramuscularly 24 hours and 4 hours prior toinfectious challenge. A defined polymicrobic infectious challenge (cecalinoculum) was placed into a gelatin capsule which was then surgicallyimplanted into the peritoneal cavity of anesthetized rats through ananterior midline incision. The early peritonitis from thisexperimentally induced infection was associated with the presence ofgram-negative organisms within the blood and peritoneal cavityculminating in mortality. The cecal inoculum contained an array offacultative species, such E. coli, as well as other obligate anaerobes(Streptococcus sp., Bacteroides sp., Clostridium perfringens,Clostridium ramosum, Peptostretococus racrus and broductus, Proteusmirabilis). The animals were observed four times per day for the first48 h and twice per day thereafter. The results are reported in Table 7.TABLE 7 Effect of Neutral Soluble Glucan on Mortality in a Rat Model forIntra-abdominal Sepsis Group Mortality (%) P vs. Saline Saline 12/20(60) Neutral soluble glucan  2/10 (10) <0.01

[0082] These results demonstrate that neutral soluble glucan which doesnot induce IL-1β and TNFα protects mice from lethal bacterial challenge.

Example 8: Demonstration Of Safety For Human Administration

[0083] A randomized, double-blind, placebo-controlled clinical trial wasconducted on healthy males to evaluate the safety of neutral solubleglucan (2.25 mg/kg) injected by intravenous infusion compared to aplacebo control. No adverse effects were observed. There was also noobserved elevation in IL-1, TNF, IL-6, IL-8 and GM-CSF. Singleintravenous administration of neutral soluble glucan resulted in anincrease in monocytes and neutrophils and in the killing activity ofthese cells proving that neutral soluble glucan retains the desirableimmunological activities in humans. See Tables 8, 9-and 10 below.However, as shown in FIGS. 5 and 6 no changes occurred in serum IL-1 andTNF and none of the patients experienced fever or inflammatoryreactions. The results are consistent with the in vitro data reported inthe earlier examples. TABLE 8 Change In Absolute Neutrophil Counts (×1000/μl) After Neutral Soluble Glucan Administration Dose Level B Hour 8Hour 12 Hour 24 Saline Mean 4.06 4.34 4.31 3.43 SD 2.12 1.53 1.16 1.46 N6 6 6 6 2.5 mg/kg Mean 4.11 11.29* 8.18 5.32 Neutral SD 1.15 4.39 3.801.75 Soluble N 6 6 6 6 Glucan

[0084] TABLE 9 Change in Monocyte Counts (× 1000/μl) After SolubleNeutral Glucan Administration Dose Level B Hour 8 Hour 12 Hour 24 SalineMean 0.33 0.44 0.59 0.33 SD 0.09 0.10 0.22 0.12 N 6 6 6 6 2.5 mg/kg Mean0.24 0.63* 0.67* 0.31 Neutral SD 0.10 0.24 0.32 0.15 Soluble N 6 6 6 6Glucan

[0085] TABLE 10 Ex Vivo Microbicidal Activity of Normal VolunteersReceiving Neutral Soluble Glucan Mean Change in % Killing¹ Dose LevelHour 3 Hour 6 Hour 24 Day 2 Day 3 Day 6 Saline 0 0 0 0 0 0 2.5 mg/kgMean 42.86 32.33 20.90 48.96 39.22 31.17 Neutral N 6 6 6 6 6 6 Solublep-Value 0.062 0.036 0.300 0.045 0.085 0.026 Glucan

Example 9: Demonstration Of Efficacy In Vivo As Human Anti-Invective

[0086] In this clinical study, the safety, tolerance, and potentialefficacy of the neutral soluble β-glucan was evaluated in patientsundergoing major thoracoabdominal surgery with high risk ofpost-operative infection. Thirty-four males and females who underwentsurgery received 0.5 mg/kg of the neutral soluble β-glucan . preparationor saline placebo, given as an intravenous infusion of 50 to 200 ml overone hour. Patients received multiple sequential doses of the neutralsoluble β-glucan or placebo at 12 to 24 hours prior to surgery, 1 to 4hours prior to surgery, 48 hours post-surgery, and 96 hourspost-surgery.

[0087] Hospitalization, infections, and usage of anti-infectivemedications were examined as potential clinical efficacy parameters.Compared to patients given saline placebo infusions, patients whoreceived the neutral soluble β-glucan spent an average of five fewerdays in the hospital (12.3±6.1 days versus 17.3±15.5 days) and threefewer days in the Intensive Care Unit (0.1±0.4 versus 3.3±6.3 days;p<0.03, one-way analysis of variance).

[0088] The number of anti-infective medication prescriptions written perstudy day following surgery was consistently higher for control patientsthan for β-glucan recipient patients. Control patients were prescribedan average of three times the number of anti-infective medications asβ-glucan recipients over the time period from surgery to discharge(p<0.005). During the Treatment and Post-Treatment Follow-up Phases, atotal of 22 culture-confirmed infections in 5 control patients and 8infections in 5 β-glucan recipient patients were identified (p<0.002).

[0089] Neutrophils (PMNs) and monocytes/macrophages (MOs) were purifiedfrom blood samples obtained at Baseline, Day 1, and Day 5 and examinedfor basal and phorbol myrisate acetate stimulated microbicidal activityagainst Staphylococcus aureus, Escherichia coli and Candida albicans.The neutral soluble β-glucan treatment generally increased the basal andphorbol-induced microbicidal activity of MOs and PENs.

Example 10: Wound Healing Effects Of Neutral Soluble Glucans

[0090] Wound healing studies were performed in a hairless mouse modelhaving full thickness wounds with and without Staphylococcus aureusinfection. Hairless SKH-1 inbred mice (6-8 weeks of age) wereanesthetized with ether and a midline 3 cm full thickness longitudinalincision was made with a number 10 scalpel blade, producing a fullthickness wound that did not penetrate the underlying fascia. Incisionswere closed using steel clips placed at 1 cm intervals.

[0091] Formulations of neutral soluble glucan in phosphate bufferedsaline were applied 30 minutes following wounding and reapplied at 24hour intervals during the seven day post-operative period. Twomicrograms of neutral soluble glucan/mouse per day was topicallyapplied. Wounds were examined daily and rank-ordered for effectivenessof formulation for enhancement of visual based wound healing. Woundswere scored for closure on a scale of 0-5, with 5 indicating the mosthealing. In one group of mice infected, the wound was treated with aculture of 10⁷ Staphylococcus aureus 30 minutes after wounding and 2 hrsprior to treatment with the neutral soluble glucan formulation.

[0092] Histological evaluation of the wound site of each test group wasmade. The dermis of the control group (untreated wound) was heavilyinfiltrated with both lymphocytes and monocytes/macrophages. However,re-epithelialization that occurred at the epidermal layer wasincomplete. The tissue section showed that the dermal tissue was weak,in that the tissue integrity was not maintained when it was sectioned.

[0093] The histology of the wounded tissue isolated from mice treatedfor three days with phosphate buffered saline containing the neutralsoluble glucan showed that there was a heavy infiltration of macrophagesand lymphocytes. Tissue integrity was good.

[0094] When topically applied to a wound, a composition of neutralsoluble glucan stimulated white blood cell entry and activity at thewound site and accelerated wound healing within the dermal layer of thewound. Furthermore, the composition effectively eliminated infectionproduced by bacterial infection (S. aureus) and prevented theprogression to sepsis. Untreated wounds progressed to sepsis.

Example 11: Stimulation Of Platelet Proliferation By Neutral SolubleGlucan

[0095] The platelet proliferation stimulatory effect of the neutralsoluble glucan was tested in an animal model system following eitherirradiation or administration of the chemotherapeutic agent cisplatin.These experiments demonstrated the unexpected platelet stimulatoryeffect.

[0096] More specifically, saline or neutral soluble glucan prepared asdescribed in Example 1 was administered to groups of 10 mice as a singleIV bolus 20 hours prior to radiation exposure. Mice were bilaterallyexposed to a total-body irradiation of 7.5-Gy. Fourteen days afterirradiation the mice were sacrificed and whole blood samples wereanalyzed for peripheral blood counts. As shown in FIG. 7, the plateletcell count from neutral soluble glucan-treated mice was increased nearly3-fold relative to saline-treated control levels.

[0097] In addition to tests on irradiated mice, cisplatin-treated micewere also tested for the effect of the neutral soluble glucan onplatelet hematopoiesis. Balbic mice were injected intravenously withcisplatin at a dose of 9.3 mg/kg through the tail vein one hour beforeinjecting either saline or the neutral soluble glucan, prepared asdescribed in Example 1, intramuscularly in a single dose of 0 (saline)or 2 mg/kg on Day 0. Platelet counts were determined before treatment(Day 0) and at 2, 4, 6, 8, and 10 days post-treatment. The results ofthis experiment are shown in FIG. 8. Each data point represents the meanand standard error of platelet counts from five mice. The statisticallysignificant differences (p<0.05) between the saline and neutral solubleglucan (2 mg/kg) are noted.

[0098] Biolocical DePosit

[0099]Saccharomyces cerevisiae strain R4 Ad was deposited on Aug. 20,1992 with the American Type Culture Collection (ATCC), 12301 ParklawnDrive, Rockville, Md., under the terms of the Budapest Treaty. Thestrain has been assigned ATCC accession number 74181. Upon issuance of apatent, this deposit will be irrevocable.

[0100] Equivalents

[0101] Those skilled in the art will recognize or be able to ascertain,using no more than routine experimentation, many equivalents to thespecific materials and components described herein. Such equivalents areintended to be encompassed in the scope of the following claims:

1. A neutral, aqueous soluble β-glucan preparation which enhances hostdefense mechanisms to infection and does not induce an inflammatoryresponse.
 2. A neutral, aqueous soluble β-glucan preparation of claim 1in which the host is a human.
 3. A neutral, aqueous soluble β-glucanpreparation of claim 1 which, when incubated for greater than 3 hours ata concentration of about 1 μg/ml with a human peripheral bloodmononuclear cell culture of about 5×10⁶ cells/ml, results in a less than2-fold increase in interleukin-1β and tumor necrosis factor-α synthesisover levels obtained following an otherwise identical incubation with abuffered solution lacking the β-glucan component.
 4. A neutral, aqueoussoluble β-glucan preparation of claim 1 which, when incubated forgreater than 3 hours at a concentration of about 1 μg/ml with anendotoxin-stimulated human peripheral blood mononuclear cell culture ofabout 5×10⁶ cells/ml, results in a less than 2-fold increase ininterleukin-1β and tumor necrosis factor-α synthesis over levelsobtained with endotoxin stimulation alone.
 5. A β-glucan preparation ofclaim 4 in which the human peripheral blood mononuclear cells arestimulated with Escherichia coli lipopolysaccharide endotoxin at aconcentration of about 1 ng/ml.
 6. A neutral, aqueous soluble β-glucanpreparation consisting essentially of a molecularspecies which migratesas a single peak when analyzed by gel permeation chromatography, themolecular species being characterized by a triple helical conformation.7. A neutral, aguecus soluble β-glucan of claim 6 wherein the molecularspecies binds specifically to the β-glucan receptor of human monocytes.8. A neutral, agueous soluble β-glucan having a triple helicalconformation which when mixed at a concentration of 1 mg/ml with anilineblue forms a fluorescent complex in 25 mM NaOE and which loses about 50%of its 25 mM NaOH fluorescence in 150 mM NaOH.
 9. A method for producinga neutral, aqueous soluble β-glucan preparation, comprising: a) treatinga suspension of insoluble β-glucan with an organic acid under conditionssufficient to dissolve the organic acid-soluble portion of the β-glucan;b) treating the organic acid-soluble β-glucan with alkali underconditions sufficient to denature the native conformation of the solubleβ-glucan; c) neutralizing the denatured soluble β-glucan underconditions sufficient to re-anneal the soluble β-glucan; and d)purifying the re-annealed soluble β-glucan to obtain a neutrals aqueoussoluble β-glucan having a triple helical conformation which, whenincubated for greater than 3 hours at a concentration of about 1 μg/mlwith an endotoxin stimulated human peripheral blood mononuclear cellculture of about 5×10⁶ cells/ml, results in a less than 2-fold increasein interleukin-1β and tumor necrosis factor-α synthesis over levelsobtained with endotoxin stimulation alone.
 10. A method of claim 9wherein the insoluble β-glucan is a whole glucan particle.
 11. A methodof claim 9 wherein step a) is performed at a pH of from about 1 to about5 and a temperature of from about 20 to about 100° C.
 12. A method ofclaim 9 wherein the organic acid is acetic acid or formic acid.
 13. Themethod of claim 9 wherein step (b) is performed at a pH of from about 7to about 14 and a temperature of from about 40 to about 121° C.
 14. Themethod of claim 9 further comprising the step of purifying the denaturedβ-glucan prior to step (c) to remove insoluble β-glucans and aggregatedsoluble β-glucans therefrom.
 15. The method of claim 9 wherein thepurification step is performed using 1,000 to 100,000 dalton nominalmolecular weight cut-off ultrafilters.
 16. The method of claim 9 whereinstep (c) is performed at a pH of about 3.5 to 11.0 and at a temperatureof from about 50 to 70° C.
 17. The method of claim 9 wherein the step(d) is performed using a 30,000 to 70,000 nominal molecular weightcut-off ultrafilter and a 100,000 to 500,000,nominal molecular weightcut-off ultrafilter.
 18. A neutral, aqueous soluble β-glucan produced bythe method of claim
 9. 19. A method for preventing infection in a mammalthat is at risk for infection, the method comprising parenterallyadministering to the mammal a neutral, aqueous soluble β-glucan which,when incubated for greater than 3 hours at a concentration of about 1μg/ml with a human peripheral blood mononuclear cell culture of about5×10⁶ cells/ml, results in a less than 2-fold increase in interleukin-1βand tumor necrosis factor-α synthesis over levels obtained following anotherwise identical incubation with a buffered solution lacking theβ-glucan component.
 20. A method of claim 19 wherein the mammal is atrisk for infection as a result of an invasive surgical procedure.
 21. Amethod for stimulating repair and healing of a wound site on a mammalcomprising administering to the wound site, an effective amount of aneutral, aqueous soluble β-glucan which, when incubated for greater than3 hours at a concentration of about 1 μg/ml with a human peripheralblood mononuclear cell culture of about 5×10⁶ cells/ml, results in aless than 2-fold increase in interleukin-1β and tumor necrosis factor-αsynthesis over levels obtained following an otherwise identicalincubation with a buffered solution lacking the β-glucan component. 22.A method of claim 21 wherein the β-glucan is topically administered tothe wound site or is injected into the wound site.
 23. A method forstimulating platelet proliferation, comprising administering to a mammala composition comprising a neutral, aqueous soluble β-glucan in aphysiologically acceptable vehicle, the neutral, aqueous solubleβ-glucan being prepared by: a) treating a suspension of insoluble,β-glucan with an organic acid under conditions sufficient to dissolvethe organic acid-soluble portion of the β-glucan; b) treating theorganic acid-soluble β-glucan with alkali under conditions sufficient todenature the native conformation of the soluble β-glucan; c)neutralizing the denatured soluble β-glucan under conditions sufficientto re-anneal the soluble β-glucan; and d) purifying the re-annealedsoluble β-glucan to obtain a neutral, aqueous soluble β-glucan having atriple helical conformation which, when incubated for greater than 3hours at a concentration of about 1 μg/ml with an endotoxin stimulatedhuman peripheral blood mononuclear cell culture of about 5×10⁶ cells/ml,results in a less than 2-fold increase in interleukin-1β and tumornecrosis factor-α synthesis over levels obtained with endotoxinstimulation alone.
 24. A pharmaceutical composition comprising aneutral, aqueous soluble β-glucan preparation which enhances hostdefense mechanisms to infection and does not induce an inflammatoryresponse, the neutral, aqueous soluble β-glucan preparation beingsolubilized in a pharmaceutically acceptable carrier.
 25. Apharmaceutical composition of claim 24 which, when incubated for greaterthan 3 hours at a concentration of about 1 μg/ml with a human peripheralblood mononuclear cell culture of about 5×10⁶ cells/ml, results in aless than 2-fold increase in interleukin-1β and tumor necrosis factorssynthesis over levels obtained following an otherwise identicalincubation with a pharmaceutically acceptable carrier lacking theβ-glucan component.
 26. A method for treating infection in a mammal thatis at risk for infection, the method comprising parenterallyadministering to the mammal a neutral, aqueous soluble β-glucan which,when incubated for greater than 3 hours at a concentration of about 1μg/ml with a human peripheral blood mononuclear cell culture of about5×10⁶ cells/ml, results in a less than 2-fold increase in interleukin-1βand tumor necrosis factor-α synthesis over levels obtained following anotherwise identical incubation with a buffered solution lacking theβ-glucan component.
 27. A method of claim 26 wherein the mammal is atrisk for infection as a result of an invasive surgical procedure. 28.The method of claim 16 wherein step (c) is performed at a ph of about6.0 to 8.0.